Summary The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce γ-H2AX. γ-H2AX stabilizes cell cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve γ-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2 and PP4R3β, that specifically dephosphorylates ATR-mediated γ-H2AX generated during DNA replication. PP4 efficiently dephosphorylates γ-H2AX within mononucleosomes in vitro. The effect of PP4 on γ-H2AX is independent of ATR and checkpoint kinase activity. When the PP4 complex is silenced, repair of DNA replication mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore γ-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto-and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G2/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (γ-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of γ-H2AX, the dissolution of IR-induced foci, and release from the G2/M checkpoint in vivo.

Microcephaly is a congenital reduction in brain size greater than three standard deviations (S.D.) below the age-related mean. Primary microcephaly (MCPH, OMIM (Online Mendelian Inheritance of Man) 251200) is further defined by the absence of significant neurological deficits, other than variable degrees of mental retardation (1). Six loci have been mapped that account for this autosomal recessive disease. The genes at two of these loci have been identified. The ASPM (abnormal spindle-like microcephaly associated) gene at the MCPH5 locus encodes the human ortholog of the Drosophila melanogaster abnormal spindle gene (asp), which is essential for normal mitotic spindle function in embryonic neuroblasts (2). The MCPH1 gene at the MCPH1 locus encodes a polypeptide with an amino-terminal and a tandem repeat of carboxyl-terminal BRCT domains (3). Recently, Mcph1 was identified as a transcriptional suppressor of the human telomerase catalytic subunit and, therefore, was termed BRIT1 (BRCT-repeat inhibitor of hTERT expression) (4).BRCT domains are peptide-and phosphopeptide-binding modules (5-7). BRCT domains are present in a number of genes involved in DNA checkpoint controls and DNA repair. These include BRCA1, 53BP1, and NFBD1/MDC1, which encode proteins with twin carboxyl-terminal BRCT domains (PTCB). The PTCBs are candidate "mediators" that may be human orthologs of the prototype budding yeast checkpoint gene RAD9. The PTCBs form discrete nuclear foci upon DNA damage. They are required for the intra-S-phase and the G 2 /M checkpoints induced by ionizing radiation (IR) 1 and regulate activation of the checkpoint kinases . Depletion of endogenous NFBD1 also impairs IR-induced ATM activation (17). The potential role of Mcph1 in IR-induced checkpoints has yet to be elucidated.In this study, we show that Mcph1 forms IR-induced foci. Inhibition of Mcph1 expression by siRNA leads to the defective IR-induced intra-S-phase and G 2 /M checkpoints and is associated with decrease of Brca1 and Chk1 both at the mRNA and protein levels. We conclude that Mcph1 is involved in DNA damage-induced cellular responses, and we propose that regulation of Brca1 and/or Chk1 by Mcph1 may contribute to these cellular responses. EXPERIMENTAL PROCEDURESPlasmids-Full-length human Mcph1 was compiled from two expressed sequence tag clones (IMAGE 6277985 and 4838618) by PCR and subcloned into the EcoRI and XhoI sites of pcDNA3HA and pcDNA3FLAG-GST vector, resulting in pcDNA-HA-Mcph1 and pcDNA-Flag-GST-Mcph1, respectively. These constructs were verified by sequence analysis. Primer sequences and detailed cloning strategies are available upon request.Northern Analysis-Probes for Northern blots correspond to nt 1861-2060 of the Mcph1 coding sequence, nt 1082-1281 of the Chk1 coding sequence, nt 5189 -5428 of the Brca1 coding sequence, and nt 778 -904 of the human ␤-actin coding sequence. These fragments were amplified from pcDNA-HA-Mcph1, pcDNA-HA-Chk1, 2 pcDNA-HA-Brca1 (a kind gift of David Livingston), and a human ␤-actin cDNA fragment (Ambion), re...

Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage (1). We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, we found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints.